Pipette Solution for Patch Clamps

2018 ◽  
Vol 2018 (4) ◽  
pp. pdb.rec103796
Keyword(s):  
Patch Clamps ◽  
Pipette Solution

Parathyroid Ca2+-conducting currents are modulated by muscarinic receptor agonists and antagonists

1997 ◽  
Vol 273 (5) ◽  
pp. E880-E890 ◽  
Author(s):  
Wenhan Chang ◽  
Tsui-Hua Chen ◽  
Stacy A. Pratt ◽  
Benedict Yen ◽  
Michael Fu ◽  
...  
Keyword(s):  
Muscarinic Receptors ◽  
Inositol Phosphate ◽  
Receptor Protein ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Pipette Solution ◽  
Pcr Products ◽  
Inositol Phosphate Accumulation ◽  
Receptor Cdna ◽  
Dose Dependent

Parathyroid cells express Ca2+-conducting cation currents, which are activated by raising the extracellular Ca2+ concentration ([Ca2+]o) and blocked by dihydropyridines. We found that acetylcholine (ACh) inhibited these currents in a reversible, dose-dependent manner (50% inhibitory concentration ≈10−8 M). The inhibitory effects could be mimicked by the agonist (+)-muscarine. The effects of ACh were blunted by the antagonist atropine and reversed by removing ATP from the pipette solution. (+)-Muscarine enhanced the adenosine 3′,5′-cyclic monophosphate (cAMP) production by 30% but had no effect on inositol phosphate accumulation in parathyroid cells. Oligonucleotide primers, based on sequences of known muscarinic receptors (M1-M5), were used in reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify receptor cDNA from parathyroid poly (A)+ RNA. RT-PCR products displayed >90% nucleotide sequence identity to human M2- and M4-receptor cDNAs. Expression of M2-receptor protein was further confirmed by immunoblotting and immunocytochemistry. Thus parathyroid cells express muscarinic receptors of M2 and possibly M4 subtypes. These receptors may couple to dihydropyridine-sensitive, cation-selective currents through the activation of adenylate cyclase and ATP-dependent pathways in these cells.

Nitric oxide mediates outward potassium currents in opossum esophageal circular smooth muscle

1996 ◽  
Vol 270 (6) ◽  
pp. G932-G938 ◽  
Author(s):  
J. Jury ◽  
K. R. Boev ◽  
E. E. Daniel
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Circular Muscle ◽  
Outward Current ◽  
Equilibrium Potential ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Circular Smooth Muscle ◽  
Outward Currents

Single smooth muscle cells from the opossum body circular muscle were isolated and whole cell currents were characterized by the whole cell patch-clamp technique. When the cells were held at -50 mV and depolarized to 70 mV in 20-mV increments, initial small inactivating inward currents were evoked (-30 to 30 mV) followed by larger sustained outward currents. Depolarization from a holding potential of -90 mV evoked an initial fast inactivating outward current sensitive to 4-aminopyridine but not to high levels of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The outward currents reversed near K+ equilibrium potential and were abolished when KCl was replaced by CsCl in the pipette solution. The sustained outward current was inhibited by quinine and cesium. High EGTA in the pipette solution reduced but did not abolish the sustained outward currents, suggesting that both Ca(2+)-dependent and -independent currents were evoked. The nitric oxide (NO)-releasing agents Sin-1 and sodium nitroprusside increased outward K+ currents. High levels of EGTA in the pipette solution abolished the increase in outward current induced by Sin-1. The presence of cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum (SR) Ca2+ pump, blocked the effects of NO-releasing agents. We conclude that NO release activates K+ outward currents in opossum esophagus circular muscle, which may depend on Ca2+ release from the SR stores.

scholarly journals Activation of Ca(2+)-dependent K+ current by acetylcholine and histamine in a human gastric epithelial cell line.

1993 ◽  
Vol 102 (4) ◽  
pp. 667-692 ◽  
Author(s):  
E Hamada ◽  
T Nakajima ◽  
S Ota ◽  
A Terano ◽  
M Omata ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Epithelial Cell ◽  
Cell Line ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Induced Response ◽  
Epithelial Cell Line ◽  
Bathing Solution ◽  
Pipette Solution ◽  
K Current

The effects of acetylcholine (ACh) and histamine (His) on the membrane potential and current were examined in JR-1 cells, a mucin-producing epithelial cell line derived from human gastric signet ring cell carcinoma. The tight-seal, whole cell clamp technique was used. The resting membrane potential, the input resistance, and the capacitance of the cells were approximately -12 mV, 1.4 G ohms, and 50 pF, respectively. Under the voltage-clamp condition, no voltage-dependent currents were evoked. ACh or His added to the bathing solution hyperpolarized the membrane by activating a time- and voltage-independent K+ current. The ACh-induced hyperpolarization and K+ current persisted, while the His response desensitized quickly (< 1 min). These effects of ACh and His were mediated predominantly by m3-muscarinic and H1-His receptors, respectively. The K+ current induced by ACh and His was inhibited by charybdotoxin, suggesting that it is a Ca(2+)-activated K+ channel current (IK.Ca). The measurement of intracellular Ca2+ ([Ca2+]i) using Indo-1 revealed that both agents increased [Ca2+]i with similar time courses as they increased IK.Ca. When EGTA in the pipette solution was increased from 0.15 to 10 mM, the induction of IK.Ca by ACh and His was abolished. Thus, both ACh and His activate IK.Ca by increasing [Ca2+]i in JR-1 cells. In the Ca(2+)-free bathing solution (0.15 mM EGTA in the pipette), ACh evoked IK.Ca transiently. Addition of Ca2+ (1.8 mM) to the bath immediately restored the sustained IK.Ca. These results suggest that the ACh response is due to at least two different mechanisms; i.e., the Ca2+ release-related initial transient activation and the Ca2+ influx-related sustained activation of IK.Ca. Probably because of desensitization, the Ca2+ influx-related component of the His response could not be identified. Intracellularly applied inositol 1,4,5-trisphosphate (IP3), with and without inositol 1,3,4,5-tetrakisphosphate (IP4), mimicked the ACh response. IP4 alone did not affect the membrane current. Under the steady effect of IP3 or IP3 plus IP4, neither ACh nor His further evoked IK.Ca. Intracellular application of heparin or of the monoclonal antibody against the IP3 receptor, mAb18A10, inhibited the ACh and His responses in a concentration-dependent fashion. Neomycin, a phospholipase C (PLC) inhibitor, also inhibited the agonist-induced response in a concentration-dependent fashion. Although neither pertussis toxin (PTX) nor N-ethylmaleimide affected the ACh or His activation of IK,Ca, GDP beta S attenuated and GTP gamma S enhanced the agonist response.(ABSTRACT TRUNCATED AT 400 WORDS)

The volume-sensitive organic osmolyte-anion channel VSOAC is regulated by nonhydrolytic ATP binding

1994 ◽  
Vol 267 (5) ◽  
pp. C1203-C1209 ◽  
Author(s):  
P. S. Jackson ◽  
R. Morrison ◽  
K. Strange
Keyword(s):  
Ketone Bodies ◽  
Anion Channel ◽  
Metabolic Inhibitors ◽  
Cell Swelling ◽  
Organic Osmolytes ◽  
Pipette Solution ◽  
Organic Osmolyte ◽  
Atp Levels ◽  
Patch Pipette ◽  
Taurine Efflux

Efflux of intracellular organic osmolytes to the external medium is a ubiquitous response to cell swelling. Accumulating evidence indicates that volume regulatory loss of structurally unrelated organic osmolytes from cells is mediated by a relatively nonselective volume-sensitive anion channel. In C6 cells, we have termed this channel VSOAC for volume-sensitive organic osmolyte-anion channel. Swelling-induced activation of VSOAC required the presence of ATP or nonhydrolyzable ATP analogues [adenosine 5'-O-(3-thiotriphosphate), adenylylmethyl-enediphosphonate (AMP-PCP), or 5'-adenylylimidodiphosphate] in the patch pipette. Sustained activation of VSOAC also required ATP. Channel rundown was observed when cellular ATP levels were lowered by intracellular dialysis with the patch pipette solution. Rundown was prevented by the ATP analogue AMP-PCP. Passive swelling-induced myo-[3H]inositol and [3H]taurine efflux was blocked by metabolic inhibitors that decreased cellular ATP levels. Titration of cellular ATP levels with azide demonstrated that the apparent dissociation constant (Kd) for ATP of both myo-inositol and taurine efflux was approximately 1.7 mM. The high Kd for ATP indicates that cellular metabolic state plays an important role in modulating organic osmolyte loss. Regulation of VSOAC activity by ATP prevents depletion of metabolically expensive organic osmolytes when cellular energy production is reduced. In addition, ATP-dependent regulation provides essential feedback to minimize the loss of energy-producing carbon sources such as pyruvate, short-chain fatty acids, ketone bodies, and amino acids, which readily permeate this channel.

scholarly journals Electrostatic Interaction of Internal Mg2+ with Membrane PIP2 Seen with KCNQ K+ Channels

2007 ◽  
Vol 130 (3) ◽  
pp. 241-256 ◽  
Author(s):  
Byung-Chang Suh ◽  
Bertil Hille
Keyword(s):  
Time Course ◽  
Inhibitory Effects ◽  
Response Curves ◽  
Pipette Solution ◽  
Current Time ◽  
Kv7 Channels ◽  
Cellular Processes ◽  
Polyvalent Cations ◽  
Voltage Dependent ◽  
Phosphatidylinositol 4

Activity of KCNQ (Kv7) channels requires binding of phosphatidylinositol 4,5-bisphosphate (PIP2) from the plasma membrane. We give evidence that Mg2+ and polyamines weaken the KCNQ channel–phospholipid interaction. Lowering internal Mg2+ augmented inward and outward KCNQ currents symmetrically, and raising Mg2+ reduced currents symmetrically. Polyvalent organic cations added to the pipette solution had similar effects. Their potency sequence followed the number of positive charges: putrescine (+2) < spermidine (+3) < spermine (+4) < neomycin (+6) < polylysine (≫+6). The inhibitory effects of Mg2+ were reversible with sequential whole-cell patching. Internal tetraethylammonium ion (TEA) gave classical voltage-dependent block of the pore with changes of the time course of K+ currents. The effect of polyvalent cations was simpler, symmetric, and without changes of current time course. Overexpression of phosphatidylinositol 4-phosphate 5-kinase Iγ to accelerate synthesis of PIP2 attenuated the sensitivity to polyvalent cations. We suggest that Mg2+ and other polycations reduce the currents by electrostatic binding to the negative charges of PIP2, competitively reducing the amount of free PIP2 available for interaction with channels. The dose–response curves could be modeled by a competition model that reduces the pool of free PIP2. This mechanism is likely to modulate many other PIP2-dependent ion channels and cellular processes.

The Neuromuscular Junction Revisited: Ca2+ Channels and Transmitter Release in Cholinergic Neurones in Xenopus Nerve and Muscle Cell Culture

1990 ◽  
Vol 153 (1) ◽  
pp. 129-140 ◽  
Author(s):  
T. P. FENG ◽  
ZHENG-SHAN DAI
Keyword(s):  
Neuromuscular Junction ◽  
Transmitter Release ◽  
Single Channel ◽  
Calcium Currents ◽  
Patch Clamps ◽  
Whole Cell ◽  
Voltage Gated ◽  
Cholinergic Neurones ◽  
Ca2 Channels ◽  
Evoked Transmitter Release

Although the entry of calcium ions into the presynaptic nerve terminals through voltage-gated Ca2+ channels is now universally recognized as playing an essential role in evoked transmitter release at the neuromuscular junction (NMJ), and indeed in chemical synapses generally, we have as yet very little direct knowledge of the Ca2+ channels of the presynaptic terminals. In this work, making use of cocultured nerve and muscle cells from Xenopus embryos, we studied the NMJ formed between the soma of identified cholinergic neurones and myoball, which allowed the use of patch-clamps on both the pre- and postsynaptic components. Both whole-cell and single-channel recordings of Ca2+ channels in the presynaptic cell were made. We found only one type of voltage-gated Ca2+ channel with highvoltage activation and slow inactivation characteristics, allowing its classification either as the L or the N type. The channels were susceptible to block by metenkephalin but not to block by nifedipine or to enhancement by Bay K 8644. This combination of pharmacological properties favours their classification as the N type. Preliminary observations on the correlation between calcium currents and transmitter release disclosed a strikingly rapid run-down of the evoked release with unchanged calcium currents and spontaneous release during whole-cell recording, indicating a specific wash-out effect on some link between calcium entry and evoked transmitter release.

GABA- and Glycine-Mimetic Responses of Linalool on the Substantia Gelatinosa of the Trigeminal Subnucleus Caudalis in Juvenile Mice: Pain Management through Linalool-Mediated Inhibitory Neurotransmission

2021 ◽  
pp. 1-12
Author(s):  
Thao Nguyen Thi Phuong ◽  
Seon Hui Jang ◽  
Santosh Rijal ◽  
Woo Kwon Jung ◽  
Junghyun Kim ◽  
...  
Keyword(s):  
Pain Management ◽  
Receptor Antagonist ◽  
Substantia Gelatinosa ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Inhibitory Neurotransmission ◽  
Wide Range ◽  
Nociceptive Responses ◽  
Inward Currents ◽  
Trigeminal Subnucleus Caudalis

Linalool, a major odorous constituent in essential oils extracted from lavender, is known to have a wide range of physiological effects on humans including pain management. The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is involved in transmission of orofacial nociceptive responses through thin myelinated A[Formula: see text] and unmyelinated C primary afferent fibers. Up to date, the orofacial antinociceptive mechanism of linalool concerning SG neurons of the Vc has not been completely clarified yet. To fill this knowledge gap, whole-cell patch-clamp technique was used in this study to examine how linalool acted on SG neurons of the Vc in mice. Under a high chloride pipette solution, non-desensitizing and repeatable linalool-induced inward currents were preserved in the presence of tetrodotoxin (a voltage-gated Na[Formula: see text]channel blocker), CNQX (a non-NMDA glutamate receptor antagonist), and DL-AP5 (an NMDA receptor antagonist). However, linalool-induced inward currents were partially suppressed by picrotoxin (a GABA[Formula: see text] receptor antagonist) or strychnine (a glycine receptor antagonist). These responses were almost blocked in the presence of picrotoxin and strychnine. It was also found that linalool exhibited potentiation with GABA- and glycine-induced responses. Taken together, these data show that linalool has GABA- and glycine-mimetic effects, suggesting that it can be a promising target molecule for orofacial pain management by activating inhibitory neurotransmission in the SG area of the Vc.

scholarly journals Spontaneous Synaptic Activity Is Primarily GABAergic in Vestibular Nucleus Neurons of the Chick Embryo

2003 ◽  
Vol 90 (2) ◽  
pp. 1182-1192 ◽  
Author(s):  
Mei Shao ◽  
June C. Hirsch ◽  
Christian Giaume ◽  
Kenna D. Peusner
Keyword(s):  
Glutamate Receptor ◽  
Vestibular Nucleus ◽  
Glycine Receptor ◽  
Brain Slices ◽  
Reversal Potential ◽  
Cell Voltage ◽  
Synaptic Activity ◽  
Pipette Solution ◽  
Principal Cells ◽  
Postsynaptic Currents

The principal cells of the chick tangential nucleus are vestibular nucleus neurons participating in the vestibular reflexes. In 16-day embryos, the application of glutamate receptor antagonists abolished the postsynaptic responses generated on vestibular-nerve stimulation, but spontaneous synaptic activity was largely unaffected. Here, spontaneous synaptic activity was characterized in principal cells from brain slices at E16 using whole cell voltage-clamp recordings. With KCl electrodes, the frequency of spontaneous inward currents was 3.1 Hz at –60 mV, and the reversal potential was +4 mV. Cs-gluconate pipette solution allowed the discrimination of glycine/GABAA versus glutamate receptor-mediated events according to their different reversal potentials. The ratio for spontaneous excitatory to inhibitory events was about 1:4. Seventy-four percent of the outward events were GABAA, whereas 26% were glycine receptor-mediated events. Both pre- and postsynaptic GABAB receptor effects were shown, with presynaptic GABAB receptors inhibiting 40% of spontaneous excitatory postsynaptic currents (sEPSCs) and 53% of spontaneous inhibitory postsynaptic currents (sIPSCs). With TTX, the frequency decreased ∼50% for EPSCs and 23% for IPSCs. These data indicate that the spontaneous synaptic activity recorded in the principal cells at E16 is primarily inhibitory, action potential-independent, and based on the activation of GABAA receptors that can be modulated by presynaptic GABAB receptors.

Phosphorylation-independent regulation of cardiac IK by guanine nucleotides and isoproterenol

1992 ◽  
Vol 262 (4) ◽  
pp. H1298-H1302 ◽  
Author(s):  
L. C. Freeman ◽  
W. M. Kwok ◽  
R. S. Kass
Keyword(s):  
G Protein ◽  
Guanine Nucleotides ◽  
Delayed Rectifier ◽  
Indirect Pathway ◽  
Beta Adrenergic ◽  
Pipette Solution ◽  
Beta Adrenergic Agonists ◽  
Ventricular Cells ◽  
Ik Channel ◽  
Inside Out

We tested for direct G protein regulation of delayed rectifier K+ (IK) channels, by measuring IK currents in guinea pig ventricular cells using patch-clamp procedures. In excised inside-out patches, IK was enhanced by adding guanosine triphosphate or guanosine 5'-O-(3-thiotriphosphate) to the cytoplasmic side, even in the presence of phosphorylation inhibitors. Enhancement of patch IK did not require extracellular agonist; however, enhancement of IK was also seen when isoproterenol was included in the pipette solution. Whole cell IK currents were increased by isoproterenol when phosphorylation pathways were blocked. These data demonstrate that guanine nucleotides and beta-adrenergic agonists can enhance IK by a phosphorylation-independent pathway. Our findings are consistent with a direct coupling of the beta-adrenergic receptor to the cardiac IK channel via a membrane-delimited G protein pathway, in addition to the well-established indirect pathway.

Sign in / Sign up

Export Citation Format

Share Document